jak2/stat2/stat3arerequiredformyogenicdifferentiation s · 2009-01-02 · immunostaining—the jak2...

JAK2/STAT2/STAT3 Are Required for Myogenic Differentiation*□S

Received for publication, April 18, 2008, and in revised form, September 16, 2008 Published, JBC Papers in Press, October 2, 2008, DOI 10.1074/jbc.M803012200

Kepeng Wang, Chihao Wang, Fang Xiao, Haixia Wang, and Zhenguo Wu1

From the Department of Biochemistry, Hong Kong University of Science & Technology, Clearwater Bay, Kowloon,Hong Kong, China

Skeletal muscle satellite cell-derived myoblasts are mainlyresponsible for postnatal muscle growth and injury-inducedregeneration. However, the cellular signaling pathways thatcontrol proliferation and differentiation of myoblasts remainpoorly defined. Recently, we found that JAK1/STAT1/STAT3not only participate in myoblast proliferation but also activelyprevent them frompremature differentiation.Unexpectedly, wefound that a related pathway consisting of JAK2, STAT2, andSTAT3 is required for early myogenic differentiation. Interfer-ence of this pathway by either a small molecule inhibitor orsmall interfering RNA inhibits myogenic differentiation. Con-sistently, all three molecules are activated upon differentiation.The pro-differentiation effect of JAK2/STAT2/STAT3 is par-tially mediated by MyoD and MEF2. Interestingly, the expres-sion of the IGF2 gene and the HGF gene is also regulated byJAK2/STAT2/STAT3, suggesting that this pathway could alsopromote differentiation by regulating signaling moleculesknown to be involved in myogenic differentiation. In summary,our current study reveals a novel role for the JAK2/STAT2/STAT3 pathway in myogenic differentiation.

Myogenic differentiation is critically dependent on two fam-ilies of transcription factors: one is the myogenic regulatoryfactor (MRF)2 family, which includes Myf5, MyoD, myogenin,and MRF4 (1–3), and the other is the myocyte enhancer factor2 (MEF2) family, which consists of MEF2A, MEF2B, MEF2C,andMEF2D (4, 5).Members of theMRF andMEF2 families canphysically interact with each other to synergistically activatemany muscle-specific genes (6). Among four MRFs, Myf5,MyoD, andMRF4 are thought to function to establish themyo-genic fate in muscle precursor cells, whereas myogenin ismainly involved in execution of the differentiation program

(7–10). When grown in culture, proliferating myoblasts do notexpress myogenin. Expression of myogenin signals that myo-blasts have irreversibly withdrawn from the cell cycle and haveinitiated the differentiation program. Both MyoD and MEF2are known to be involved inmyogenin induction at the start ofdifferentiation (11–13).Skeletal muscle satellite cells, which are a group of quiescent

cells located between basal lamina and plasma membrane ofmyofibers in mature muscles, are mainly responsible for post-natal muscle growth and injury-induced muscle regeneration.Upon muscle damage by trauma or genetic disorders, the qui-escent satellite cells are activated to re-enter the cell cycle,actively proliferate, and eventually differentiate into multi-nu-cleated myotubes to repair the damaged muscles (14–17). Dif-ferent molecular markers have been identified that can be usedto characterize muscle satellite cells at different stages. Forexample, the quiescent muscle satellite cells are known toexpress Pax7, the activated satellite cells, and proliferatingmyoblasts tend to co-express Pax7 andMyoD, whereas the dif-ferentiating myocytes start to express myogenin with concom-itant down-regulation of Pax7 (14, 15).Multiple signaling pathways and growth factors have been

found to play roles in myogenic differentiation. We and otherspreviously showed that the p38 mitogen-activated proteinkinase (MAPK) pathway is required for myogenic differentia-tion (18–20), whereas the extracellular signal-regulated kinase(ERK) pathway plays dual roles: it inhibits differentiation at theonset of differentiation and promotesmyocyte fusion at the latestage of differentiation (18, 21, 22). Insulin-like growth factors(IGFs) potently promotemyogenic differentiation via phospha-tidylinositol 3-kinase (PI3K) and Akt (22–29), whereas hepato-cyte growth factor (HGF) can stimulate myoblast proliferationand inhibit differentiation, presumably via the ERK pathway(30–33).The Janus kinase (JAK)/signal transducer and activator of

transcription (STAT) signaling pathway has been studiedextensively in cytokine signaling during blood developmentand immune responses (34). The JAK family of tyrosine kinaseshas four members: JAK1, 2, and 3 and Tyk2, whereas the STATfamily of transcription factors consists of seven members:STAT1–4, 5a, 5b, and 6. Among members of the JAK andSTAT families, STAT3 was the first to be implicated in myo-blast proliferation in muscle satellite cell-derived C2C12 cells(35, 36). Subsequently, active STAT3was also found to be pres-ent in activated muscle satellite cells and proliferating myo-blasts in regenerating rat muscles (37). In addition, STAT3 wasalso shown to physically interact withMyoD (38). Based on theeffect of AG490, an inhibitor for JAK2 (39), JAK2 was proposed

* This work was supported by Hong Kong Research Grant Council Grants663007 and CA06/07.SC02 (to Z. W.) and Area of Excellence Scheme AoE/B-15/01 from the Hong Kong University Grant Committee. The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. S1.

1 To whom correspondence should be addressed. Tel.: 852-2358-8704; Fax:852-2358-1552; E-mail: [email protected].

2 The abbreviations used are: MRF, myogenic regulatory factor; STAT, signaltransducers and activators of transcription; siRNA, small interfering RNA;MEF, myocyte enhancer factor; ERK, extracellular signal-regulated kinase;IGF, insulin-like growth factor; PI3K, phosphatidylinositol 3-kinase; HGF,hepatocyte growth factor; JAK, Janus kinase; LIF, leukemia inhibitory fac-tor; DMEM, Dulbecco’s modified Eagle’s medium; GM, growth medium;DM, differentiation medium; EGFP, enhanced green fluorescent protein;WCE, whole cell extract; RT, reverse transcription; MHC, myosin heavychain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 49, pp. 34029 –34036, December 5, 2008© 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

DECEMBER 5, 2008 • VOLUME 283 • NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 34029

http://www.jbc.org/cgi/content/full/M803012200/DC1

to be required for leukemia inhibitory factor (LIF)-inducedC2C12 proliferation (36). However, this result has to be viewedwith caution because of potential nonspecific effects of the drug(i.e. inhibition of cellularmolecules other than JAK2). Recently,our group showed that a pathway consisting of JAK1/STAT1/STAT3 is involved in myoblast proliferation (40). Perturbationof this pathway by siRNAs up-regulates the expression levels ofp21cip1 and p27kip1. Furthermore, we found that this pathwayalso inhibits the differentiation program by repressing theexpression ofMyoD andMEF2 genes, whose protein productsare required formyogenin induction at the onset of differentia-tion (11–13, 40). Down-regulation of either JAK1 or STAT1 bysiRNA accelerates myogenic differentiation in both C2C12cells and primary myoblasts (40).Although JAK2 is quite homologous to JAK1, unexpectedly,

we found that JAK2 plays an opposite role during myogenicdifferentiation in comparison with JAK1. STAT2 and STAT3appear to function downstream of JAK2 during myogenic dif-ferentiation. In addition to MyoD and MEF2, we showed thatJAK2/STAT2/STAT3 also control the expression of HGF andIGF2, both of which are known to regulate the proliferation anddifferentiation of myoblasts. This finding provides additionalmechanisms to explain why JAK2 is required for myogenicdifferentiation.

EXPERIMENTAL PROCEDURES

Isolation and Culturing of Primary Myoblasts—Isolation ofprimarymyoblasts was carried out as described previously (57).Briefly, C57/B6 mice were sacrificed by cervical dislocation.Skeletal muscles from hind limbs and back were dissected outand minced with scissors. The muscles were then digested by 1mg/ml Pronase (Calbiochem, San Diego, CA) in phosphate-buffered saline at 37 °C for 1 h to dissociatemuscle satellite cellsfrommyofibers. After digestion,myofiberswere allowed to sed-iment, and satellite cells in the supernatant were passedthrough 40-�mcell sieves (BDBiosciences, Franklin Lakes, NJ).The cells were then subjected to centrifugation in a Percoll(Sigma-Aldrich) gradient. The satellite cells were collected inthe interphase between 60 and 20% of Percoll and cultured onMatrigel (Clontech, Mountain View, CA)-coated plates. Thecells were first grown for 24 h in the plating medium (DMEMwith 10% horse serum, 0.5% chicken embryo extract, 1% peni-cillin, and streptomycin) to facilitate their attachment to plates,followed by growth in the satellite growth medium (DMEMwith 20% fetal bovine serum, 2% chicken embryo extract, 1%penicillin, and streptomycin) to promote myoblast prolifera-tion. Themyoblastswere induced to differentiate in the satellitedifferentiationmedium (DMEMwith 5% horse serum, 1% pen-icillin, and streptomycin).Cell Lines, DNAConstructs, and Reagents—C2C12 cells were

maintained in the growthmedium (GM, DMEMwith 20% fetalbovine serum, 1% penicillin, and streptomycin). To induce dif-ferentiation, the cells were grown in DMEM containing 2%horse serum (also called differentiation medium (DM)). Thecells were kept in a 37 °C incubator with 5% CO2. JAK andSTAT constructs were gifts from Dr. Zilong Wen (Hong KongUniversity of Science & Technology). JAK1 and 2, Tyk2, andSTAT1, 2, and 3 were subcloned into pcDNA3 vector with an

N-terminal FLAG tag. G133-luc, MCK-luc, 4RE-luc, 3MEF2-luc, Gal4-MyoD, Gal4-MEF2A, Gal4-MEF2C, and Gal4-MEF2D were described previously (18, 24). AG490 was pur-chased from LC Laboratories (Woburn, MA). Recombinantmouse IGF1 was from R & D (Minneapolis, MN).siRNA and Plasmid Transfection—For siRNA transfection,

50% confluent C2C12 cells or primary myoblasts were trans-fected with 100 nM siRNA using Lipofectamine 2000 (Invitro-gen). The following siRNAs (top strand sequence is shown)were used: JAK2(1) (5�-gca aac cag gaa ugc uca a), JAK2(2) (5�-gga aug gcc ugc cuu aca a), JAK2-scrambled (5�-guc aag aac cgccaa gau a), STAT2(1) (5�-gga cug guu ggc cga uua a), STAT2(2)(5�-gaa gug aau gca gag cuc uug uua g), STAT2-scrambled (5�-gug acu cag ugc gaa gug u), STAT3(1) (5�-uau cau cga ccu ugugaa a), STAT3(2) (5�-cca acg acc ugc agc aau a), STAT3-scram-bled (5�-gcc uac cgc acc aag aua a), and enhanced green fluores-cent protein (EGFP) (5�-gcu gac ccu gaa guu cau c). For plas-mids and siRNA co-transfection, 1 �g of a plasmid wasdelivered into cells in each 35-mm well with 100 mM siRNAusing Lipofectamine 2000.Luciferase Assay—For luciferase assays, the cells were first

transfected and incubated in GM for 24 h, followed by growthin DM for another 24 h. The cells were then harvested in thelysis buffer (50 mM Hepes, pH 7.6, 1% (v/v) Triton X-100, 150mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 100 mM NaF, 20 mMp-nitrophenylphosphate, 20 mM �-glycerophosphate, 50 �Msodium vanadate, 2 mM dithiothreitol, 0.5 mM phenylmethyl-sulfonyl fluoride, 2 �g/ml aprotinin, 0.5 �g/ml leupeptin, and0.7 �g/ml pepstatin) at 4 °C for 10 min. Whole cell extracts(WCEs)were obtained after centrifugation followed by removalof the debris. The luciferase activity was determined in a lumi-nometor (Berthold Technologies, Bad Wilbad, Germany) andnormalized to the amount of proteins present in each sample.The protein concentration of each sample was determined byprotein assay reagent from Bio-Rad.RT-PCR—For semi-quantitative RT-PCR, total RNAwas iso-

lated using TRIzol reagent (Invitrogen) according to the man-ufacturer’s instructions. 1 �g of total RNA from each samplewas reverse transcribed into cDNA using the ImProm-IIreverse transcription system (Promega, Madison, WI). Theexpression levels of various genes were then detected by PCR.The following primers were used for detection of gene expres-sion: myogenin (forward, 5�-gac tcc cca ctc ccc att cac ata;reverse, 5�-ggc ggc agc ttt aca aac aac aca), GAPDH (forward,5�-tga tgc tgg tgc tga gta tgt cgt; reverse, 5�-tcc ttg gag gcc atg taggcc at), MyoD (forward, 5�-agg ctc tgc tgc gcg acc; reverse,5�-tgc agt cga tct ctc aaa gca cc);MEF2A (forward, 5�-tga cct gtctgc cct gca; reverse, 5�-tta ggt cac cca tgt gtc), MEF2C (forward,5�-tga cat ccg gtg cag gca c; reverse, 5�-gag tgc tag tgc aag ctc),IGF2 (forward, 5�-tgc tgc atc gct gct tac; reverse, 5�-tga ttc actgat ggt tgc tg), and HGF (forward, 5�-gac tat gaa gct tgg ctt g;reverse, 5�-ctc aca tgg tcc tga tcc).Antibodies, Immunoblotting, Immunoprecipitation, and

Immunostaining—The JAK2 and STAT2 antisera were gener-ated by injecting rabbits with the following protein fragments:JAK2, amino acids 820–1129; STAT2, amino acids 315–568.Antibodies from serum were first affinity-purified by incubat-ing with polyvinylidene difluoride membranes containing

JAK2/STAT2/STAT3 Promote Differentiation

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transferred antigens of JAK2 or STAT2 followed by washingand elusion. The antibodies were subsequently incubated withmembranes transferred with the corresponding regions ofJAK1 or STAT1 to eliminate those that cross-react with otherfamily members. The sources of commercial antibodies used inthis work were listed as follows: anti-phospho-tyrosine(05-321), anti-STAT1 (06-501) and anti-STAT2 (07-140) werefrom Millipore (Billerica, MA); anti-myogenin (SC-12732),anti-MyoD (SC-760), anti-MEF2 (SC-313), anti-�-actin (SC-8432), and anti-Gal4-DBD (SC-510) were from Santa Cruz(Santa Cruz, CA); anti-�-tubulin (T4026) and anti-Flag (F3165)were from Sigma-Aldrich; anti-myosin heavy chain (MHC)(MF20) was from Developmental Studies Hybridoma Bank(Iowa City, IA); anti- Ser(P)473-Akt (9271), anti-Thr(P)308-Akt(9275), anti-Akt (9272), anti-Thr(P)202, Tyr(P)204-ERK (9101),anti-ERK (9102), anti-STAT3 (9132), anti-Tyr(P)705-STAT3(9131), and anti-JAK1 (3332) were from Cell Signaling Tech-nology (Danvers, MA). Immunoblotting was carried outaccording to standard procedures. To perform immuno-staining, primary myoblasts or cultured C2C12 cells were firstfixed with 4% paraformaldehyde in phosphate-buffered salinefor 10 min and then permeabilized with 0.2% Triton in phos-phate-buffered saline for 10min at room temperature, followedby incubation with primary antibodies against either myogenin(1:10) or MHC (1:10) overnight. The cells were then incubated

with appropriate secondary anti-bodies for 1 h, followed by incuba-tion with 4�,6�-diamino-2-phenyl-indole for 10 min to counterstainthe nuclei. The images were takenby a CCD camera (Spot RT; Diag-nostic Instruments Inc., SterlingHeights, MI) linked to an OlympusIX70 fluorescent microscope.Immunoprecipitation was generallyperformed by adding 1 �g of anti-body to 500 �g of whole cell lysatestogether with 20 �l (bed volume) ofprotein A-Sepharose beads. Themixture was rotated for 2 h in 4 °Cfollowed by extensive washing withthe lysis buffer.Statistics—For statistical analysis,

the p valuewas calculated using Stu-dent’s t test with p� 0.05 being con-sidered statistically significant.

RESULTS

Inhibition of JAK2 with AG490Inhibits Myogenic Differentiation—To evaluate the role of JAK2 inmyogenic differentiation, we firsttreated C2C12 cells with AG490, aknown inhibitor of JAK2, at thetime when cells were induced to dif-ferentiate. Even by phase contrastmicroscopy, we could clearly seethat the formation of multinucle-

ated myotubes was inhibited by AG490 in a dose-dependentmanner (Fig. 1A, bottom panels). Consistently, we found thatthe expression of myogenin and MHC, two proteins that arepresent at the early and late stages of differentiation, respec-tively, and are commonly used as stage-specific differentiationmarkers, was significantly inhibited by AG490 as judged byboth immunostaining and immunoblotting analysis with anti-bodies specific formyogenin andMHC (Fig. 1A, top panels). Tofind out whether AG490 has a similar inhibitory effect on pri-mary myoblasts, we applied AG490 to primary myoblastsundergoing differentiation. As shown in Fig. 1C, the differenti-ation of primary myoblasts was significantly inhibited as evi-denced by reduced myotube formation.Knockdown of JAK2 by siRNA RepressesMyogenic Differen-

tiation—Although it was convenient to employ pharmacolog-ical inhibitors like AG490 in our initial studies, it was necessaryto complement and verify the drug study with more specificassays because many pharmacological inhibitors may inhibitsomeunknowncellular targets in addition to their intended andwell characterized targets. To this end, we designed two differ-ent JAK2-specific siRNAs and two control siRNAs (i.e. anEGFP-specific siRNA and a scrambled JAK2-siRNA). C2C12cells were first transfected with individual siRNA. The cellswere then either grown in theGMor induced to differentiate inthe DM. By Western blotting, we showed that both JAK2-

FIGURE 1. AG490 inhibits myogenic differentiation in both C2C12 and primary myoblasts. Nearly conflu-ent C2C12 cells (A and B) or primary myoblasts (C) were induced to differentiate in DM with either dimethylsulfoxide (DMSO, vehicle) or different doses of AG490. A, after growing in DM for 24 h (left three panels) or 48 h(right three panels), the cells were fixed and subjected to immunostaining using antibodies against eithermyogenin or MHC. B, equal amount of WCEs were subjected to immunoblotting to reveal the expression levelsof myogenin, MHC, and �-actin (loading control). Con, dimethyl sulfoxide control. C, after growing in DM for30 h, the primary myoblasts were fixed and subjected to immunostaining for MHC. In A and C, 4�,6�-diamino-2-phenylindole (DAPI) was added to counterstain the nuclei.



siRNAs,butnot thetwocontrol siRNAs,werespecificandeffective inknocking down JAK2 gene expression without affecting the expres-sion of JAK1 gene (Fig. 2A and supplemental Fig. S1B). Importantly,both JAK2-siRNAs, but not the control siRNAs, significantly inhib-ited myogenic differentiation, as judged by reduced expression ofmyogeninandMHCinimmunoblottinganalysis (Fig.2Aandsupple-mental Fig. S1D).Toavoidunnecessarydataduplication, the effect ofone JAK2-specific siRNAwas shown in subsequent assays. By semi-quantitative RT-PCR, we showed that the JAK2-siRNA inhib-ited the expression ofmyogeninmRNA (Fig. 2B). Consistently,by reporter assays, we found that the JAK2-siRNA significantlyinhibited the expression of a luciferase reporter gene driven bya 133-bp proximal myogenin promoter (i.e. G133-Luc) (24)(Fig. 2C). By immunostaining, we also confirmed that theexpression of myogenin and MHC was inhibited by the JAK2-siRNA (Fig. 2D). Furthermore, we showed that the JAK2-siRNA also inhibited the differentiation of primary myoblasts,

as evidenced by reduced number ofmultinucleated myotubes, espe-cially those with four ormore nuclei(Fig. 2E).JAK2 Is Required for Both the

Expression and TransactivationFunction of MyoD and MEF2—Toexplain at themolecular level why theinterference of the JAK2 function byeither AG490 or siRNA repressesmyogenic differentiation, we exam-ined the status of MyoD and MEF2,because both are indispensable formyogenic differentiation and myo-genic gene expression. As shown inFig. 3A, compared with nontreatedcontrol cells, the AG490-treatedcells had significantly reduced levelsof MyoD and MEF2. Similarly, thecells treated with the JAK2-siRNAalso had reduced levels of MyoDandMEF2 when compared with thecontrol cells transfected with theEGFP-siRNA (Fig. 3B). As a control,we found that the levels of either�-actin or JAK1were not affected bythe JAK2-siRNA. Consistently,both MRF- and MEF2-dependentreporter genes (i.e. 4RE-luc and3MEF2-luc respectively) displayedobviously reduced activities whencells were co-transfected with theJAK2-siRNA (Fig. 3C). By RT-PCR,we further determined that theJAK2-siRNA-mediated reduction inMyoD and MEF2 protein levels wasmainly due to a reduction in theirmRNA levels (Fig. 3D). This sug-gested that JAK2 normally regulatesMyoD andMEF2 gene expression atthe transcription level. In addition,

we also testedwhether the transcriptional activity ofMyoD andMEF2 was affected by the JAK2-siRNA. To do this, we firstfusedMyoD or severalMEF2 genes to a cDNA fragment encod-ing the DNA-binding domain (i.e. amino acids 1–147) of yeastGal4. When these constructs were co-transfected into C2C12cells with a luciferase reporter gene under the control of multi-ple Gal4-binding sites (i.e. Gal4-luc), the reporter gene expres-sion was mainly determined by the transactivation domain ofMyoD orMEF2 (41). Interestingly, in the presence of the JAK2-siRNA, the reporter activity was considerably reduced com-pared with that in the presence of the EGFP-siRNA (Fig. 3E).This suggests that JAK2 also regulates the transcriptional activ-ity of both MyoD and MEF2, in addition to its effect on thetranscription ofMyoD andMEF2 genes.STAT2 and STAT3 Act Downstream of JAK2 to Regulate

Myogenic Differentiation—STAT proteins are the best charac-terized downstream targets of JAKs. Of the seven STAT family

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FIGURE 2. Knockdown of JAK2 by siRNA inhibits myogenic differentiation. 50% confluent C2C12 cells (A–D)or primary myoblasts (E) were transfected with either an EGFP-siRNA (control) or JAK2-siRNAs as indicatedfollowed by growth in GM for 24 h. Nearly confluent cells were induced to differentiate in DM for various timesas indicated. A, WCEs were subjected to immunoblotting by various antibodies. n.s., nonspecific. B, after cellharvest, total RNA was extracted. The mRNA levels of myogenin were determined by semi-quantitative RT-PCR.GAPDH served as a loading control. C, C2C12 cells were co-transfected with siRNAs and G133-luc. After growthin GM for 24 h and DM for 24 h, the cells were harvested, and WCE were subjected to luciferase assays. Foldchange was calculated as the ratio of the luciferase activity of cells transfected with the JAK2-siRNA overthat with the EGFP-siRNA. D, C2C12 cells were fixed and subjected to immunostaining for either myogenin(left two columns, DM36 h) or MHC (right two columns, DM48 h). E, primary myoblasts were fixed andsubjected to immunostaining for MHC. In D and E, 4�,6�-diamino-2-phenylindole (DAPI) was added tocounterstain the nuclei. Among MHC-positive myotubes in three randomly chosen fields (10� magnifi-cation), those with 2–3, 4 –7, and �8 nuclei were separately counted. The data are presented as themeans � S.D. *, p � 0.05. **, p � 0.01.






members, we recently demonstrated that STAT1 functionsdownstream of JAK1 to promote proliferation and to inhibitpremature differentiation of myoblasts (40). To identify theSTATs that function downstream of JAK2 in myogenic differ-entiation, we first designed siRNAs specifically targeting differ-ent STATs. We found that knockdown of STAT2 and STAT3consistently inhibited myogenic differentiation, and wedecided to focus on these two STATs. As shown in Fig. 4A, twodifferent siRNAs targeting either STAT2 or STAT3 were botheffective and specific. Both STAT2-siRNAs and STAT3-siRNAs considerably inhibited myogenic differentiation inC2C12 cells as judged by reduced expression of myogenin andMHC. Apart from the EGFP-siRNA, we also used scrambledSTAT2or STAT3 siRNAas another negative control. As shownin the supplemental Fig. S1E, the scrambled siRNAs had noobvious effect on the expression of STAT2 or STAT3 genes.Consequently, they did not affect the expression ofmyogenin orMHC genes. This further proved that the myogenic effectscaused by STAT2 or STAT3 siRNAs were specific. In addition,knockdown of STAT2 or STAT3 also resulted in reduced levels

of MEF2A and MEF2C mRNA, asjudged byRT-PCR (Fig. 4B). Consis-tently, both STAT2- and STAT3-siRNAs specifically inhibited theexpression of several myogenicreporter genes including G133-lucand 3MEF2-luc (Fig. 4C). Becausethe effect of both STAT2-siRNAand STAT3-siRNA was very similarto that of the JAK2-siRNA, our datasuggested that STAT2 and STAT3function downstream of JAK2 toregulate myogenic differentiation.JAK2, STAT2, and STAT3 Are

Activated upon Myogenic Differen-tiation—Because individual knock-down of the endogenous JAK2,STAT2, and STAT3 all inhibitedmyogenic differentiation (Figs. 2and 4), this suggested that JAK2,STAT2, and STAT3 are normallyactivated during myogenic differen-tiation. To test whether this is thecase, we examined the expressionlevels and the activation status ofthese three proteins in C2C12 cellsundergoing differentiation. How-ever, except for the antibodiesagainst STAT3, we found that manycommercial antibodies for JAK2and STAT2 cross-reacted withother members of the JAK/STATfamily and failed to specifically pulldown JAK2 and STAT2 in immuno-precipitation assays.3 To overcomethe problem, we generated our ownantibodies against JAK2 andSTAT2, purified them, and proved

that they were specific and suitable for immunoblottingand immunoprecipitation assays (supplemental Fig. S1,A,C, E,and F). As indicated in Fig. 5, our immunoblotting assaysrevealed that the total protein levels of JAK2, STAT2, andSTAT3 gradually increased during differentiation. As to thelevels of the tyrosine-phosphorylated forms of JAK2, STAT2,and STAT3 that represent the active forms of these proteins(34), we found that it was easy to detect the tyrosine-phospho-rylated (i.e. active) STAT3 in direct immunoblotting assaysbecause of its abundance in C2C12 cells. Unlike STAT3, theactive JAK2 and STAT2 were much more difficult to detectbecause of their low abundance and a lack of specific antibodiesrecognizing the active JAK2 and STAT2. We had to enrichthem first by immunoprecipitation followed by immunoblot-ting with a well characterized anti-phospho-tyrosine antibody(i.e. 4G10) (42). By doing so, we found that the levels of theactive JAK2 and STAT2 also increased upon differentiation, aswas the case for STAT3.

3 K. Wang and Z. Wu, unpublished data.

FIGURE 3. Inhibition of JAK2 reduces both the expression levels of MyoD and MEF2 and their transacti-vating activities. A, C2C12 cells were induced to differentiate for 24 and 48 h in the presence or absence ofdifferent doses of AG490. B and C, C2C12 cells were transfected with either the EGFP- or JAK2-siRNA followedby growth in GM for 24 h. The cells were then induced to differentiate for various times as indicated. For A andB, WCEs were subjected to immunoblotting by various antibodies. n.s., nonspecific. For D, mRNA levels ofMyoD, MEF2A, and MEF2C were determined by RT-PCR and normalized to that of GAPDH. C and E, C2C12 cellswere transfected with various reporter constructs together with either the EGFP- or JAK2-siRNAs followed bygrowth in GM for 24 h. For C, the cells were induced to differentiate for 18 h before harvest. WCEs weresubjected to luciferase assays, and the fold change was determined the same way as that described in thelegend for Fig. 2C. For E, after luciferase assays, the remaining WCEs were subjected to immunoblotting toreveal the levels of Gal4 fusion proteins in cells transfected with either the EGFP- or JAK2-siRNA.





JAK2/STAT2/STAT3 Regulate the Expression of IGF2 andHGF—Multiple intracellular signaling pathways have beenimplicated in the process ofmyogenic differentiation, includingthe IGF/PI3K/Akt pathway and the p38 MAPK pathway thatpromote differentiation and the ERK pathway that inhibits theearly differentiation (18, 19, 21, 22, 24, 43, 44). Thus, it would beinteresting to test whether modulation of JAK2/STAT2/STAT3 affects these well characterized pathways. We foundthat knockdown of JAK2 in C2C12 cells increased the levels ofthe active ERK (i.e. p-ERK) both before and at the early stage ofdifferentiation (Fig. 6A, lanes 2 and 4). In contrast, JAK2 knock-down significantly reduced the levels of the active Aktmainly atthe late stage of differentiation (Fig. 6A, lanes 6 and 8). As acontrol, the JAK2-siRNA did not affect the expression of �-tu-bulin and JAK1. Similarly, when C2C12 cells were individuallytransfected with siRNAs against STAT2 or STAT3, we foundthat the STAT2-siRNA hadmore obvious effect on the levels ofthe active ERK during early differentiation (Fig. 6B), whereasboth STAT2- and STAT3-siRNAs affected the levels of theactive Akt during late differentiation (Fig. 6C). To understandwhat caused such changes in the levels of the active Akt andERK, we examined the mRNA levels of IGF2 and HGF, both ofwhich are known to be capable of activating Akt and ERK,respectively, in myogenic cells. As shown in Fig. 6D, knock-

down of JAK2 induced HGF expression even in proliferatingmyoblasts but repressed IGF2 expression at the late stage ofdifferentiation (e.g. DM24 and DM48 h). Consistent with theirdifferential effects on the status of the active ERK and Akt (Fig.6, B and C), knockdown of STAT2 preferentially induced HGFexpression during early differentiation, whereas that of eitherSTAT2 or STAT3 repressed IGF2 expression during late differ-entiation (Fig. 6E). We further demonstrated that Akt phos-phorylation induced by the exogenous IGF was not affected bythe JAK2-siRNA (Fig. 6F), suggesting that the decreased levelsof the active Akt by JAK2-siRNA is not due to direct interrup-tion of the IGF/PI3K/Akt pathway. Rather, our data suggestedthat the JAK2/STAT2/STAT3 pathway regulates the expres-sion of both IGF2 and HGF, which in turn affects the status ofthe active Akt and ERK, respectively.

DISCUSSION

Multiple JAK/STAT Pathways Regulate Myogenic Differ-entiation with Opposing Effects—Previous studies by severalgroups have shown that several JAK and STAT members areactivated in both LIF-treated proliferating myoblasts andregenerating muscles (35–37). However, it was unclear howthese JAKs and STATs function during myogenic differenti-ation. Recently, we carried out detailed analysis in bothimmortalized C2C12 cells and primary myoblasts. We foundthat JAK1, acting via STAT1 and STAT3, is involved in myo-blast proliferation (40). Concomitantly, the JAK1/STAT1pathway represses differentiation process to prevent prema-ture differentiation of myoblasts. They do so by regulatingthe expression of MyoD, MEF2, Id1, p21cip1, and p27kip1,all of which are key players in myogenic differentiation (40).Unexpectedly, we find in this report that JAK2, a close rela-tive of JAK1, plays a positive role in myogenic differentia-tion. Because of embryonic lethality in JAK2 knock-out mice(45, 46), we could not directly analyze the myogenic role of

FIGURE 4. Knockdown of either STAT2 or STAT3 inhibits myogenic differ-entiation. C2C12 cells were transfected with either siRNAs alone (A and B) orsiRNAs plus reporter constructs (C) followed by growth in GM for 24 h. Thecells were then induced to differentiate for various times as indicated. A, WCEswere subjected to immunoblotting by various antibodies. #1 and #2 denotedifferent sets of siRNA. B, the mRNA levels of myogenin, MEF2A and MEF2Cwere determined by RT-PCR and normalized to that of GAPDH. C, cells wereinduced to differentiate for 24 h before harvest followed by luciferase assays.The fold change was determined the same way as that described in the leg-end for Fig. 2C.

FIGURE 5. JAK2, STAT2 and STAT3 are activated during myogenic differ-entiation. Near confluent C2C12 cells were induced to differentiate for vari-ous times as indicated. WCEs were subjected to immunoblotting by variousantibodies as indicated. To determine the levels of P-JAK2 and P-STAT2,immunoprecipitation (IP) was first performed using our homemade JAK2 andSTAT2 antibodies followed by immunoblotting with an antibody againstphospho-tyrosine (P-Y).



JAK2 in mice. Instead, we focused on myogenic cell cultures.Interference of the JAK2 function either by a small-moleculeinhibitor (i.e. AG490) or by JAK2-siRNA inhibits myogenicdifferentiation in both C2C12 and primary myoblasts (Figs. 1and 2). We further suggest that the pro-differentiation effectof JAK2 is mediated by STAT2 and STAT3, because theknockdown of either STAT2 or STAT3 partially recapitu-lates the effects generated by that of JAK2. In addition, bothSTAT2 and STAT3 are increasingly activated upon differen-tiation, which parallels the changes in the activity of JAK2(Fig. 5). Like the JAK1/STAT1/STAT3 pathway, the JAK2/STAT2/STAT3 pathway also regulates the expression ofMyoD and MEF2. Consistent with their opposing effects ondifferentiation, these two JAK/STAT pathways regulate theexpression of MyoD and MEF2 in opposite manners:whereas the JAK1/STAT1/STAT3 pathway represses theexpression of MyoD and MEF2 (40), the JAK2/STAT2/STAT3 pathway enhances their expression. It remainsunclear how different STAT complexes can bring aboutopposite effects on the same set of target genes. Presumably,they can do so by cooperating with different co-factors andact at different stages in the course of differentiation.Although we show that LIF activates the JAK1/STAT1/STAT3 pathway (40), we do not know at present which

ligand engages the JAK2/STAT2/STAT3 pathway during myogenicdifferentiation.STAT3 Functions atMultiple Steps

during Myogenic Differentiation—STAT3 is arguably the most impor-tant STAT during mouse embryodevelopment, because only the lossof STAT3 causes embryonic lethal-ity (47). Among many diverse rolesrevealed for STAT3, its most prom-inent role is in cell proliferation. It iswell established that STAT3 is anessential mediator downstream ofLIF in maintaining self-renewal andpluripotency of ES cells (48, 49). Inaddition to cytokines, many growthfactors including IGF and epidermalgrowth factor also result in STAT3phosphorylation and activation (50,51). STAT3 has also been linked touncontrolled cell growth based onthe following evidence: a constitu-tively activated STAT3 is capable oftransforming fibroblasts (52). Acti-vated STAT3 is frequently detectedin various tumors (53). In addition,STAT3 can also be activated by var-ious oncogenes such as Src (54). Inmyogenic cells, activated STAT3has been detected in LIF-treatedproliferating myoblasts and regen-erating muscles (35–37). In addi-tion, knockdown of STAT3 reduced

the LIF-induced myoblast proliferation (40). These data areconsistent with the role of STAT3 in proliferation.Consistent with our previous notion and a recent report (40,

55), we find that STAT3 is also required for myogenic differen-tiation. This is supported by the following results: First, STAT3is significantly activated during differentiation (Fig. 5). Second,knockdown of STAT3 inhibits myogenic differentiation (Fig. 4)(55). Our data suggest that STAT3 can function at both prolif-eration and differentiation steps presumably by associatingwith different co-factors that facilitate its differential binding todifferent subset of target genes. Further confirmation of thedual role of STAT3 in vivo awaits the generation of satellitecell/myoblast-specific STAT3 knock-out mice.JAK2/STAT2/STAT3 Regulate Expression of IGF2 and HGF—

In addition to MyoD and MEF2, we show that the JAK2/STAT2/STAT3 pathway could also regulate the expression ofHGF and IGF2; it represses HGF at the early stage of differen-tiation and enhances IGF2 at the late stage of differentiation.BothHGFand IGF2 are secreted growth factors known to influ-ence proliferation and differentiation of myoblasts (26, 27,29–32). HGF exerts its biological effect through c-Met, amem-ber of the receptor tyrosine kinase family, with ERK being amain intracellular pathway activated by HGF (33, 56), whereasIGF exerts its effect through IGF receptor, also amember of the

FIGURE 6. JAK2/STAT2/STAT3 regulate the expression of IGF2 and HGF at distinct phases of differentia-tion. C2C12 cells were transfected with different siRNAs as indicated. The cells were induced to differentiate forvarious times. For F, after growth in GM for 24 h, the cells were starved in serum-free DMEM for 1 h and thentreated with or without 50 ng/ml IGF1 for 15 min. For A–C and F, WCEs were subjected to immunoblotting withvarious antibodies as indicated. For D and E, the mRNA levels of myogenin, HGF, and IGF2 were determined byRT-PCR and normalized to that of GAPDH.



receptor tyrosine kinase family, with PI3K/Akt being the mainpathway activated by IGF in myogenic cells (22–24, 29). Con-sistent with a positive role for HGF in cell proliferation and anegative role in early differentiation (30–32), down-regulationofHGF gene by JAK2 or STAT2 at the early stage of differenti-ation facilitates myogenic differentiation. Because IGF2 is anautocrine factor known to promote differentiation (26, 27, 29),an induction of IGF2 by JAK2, STAT2, or STAT3 during latedifferentiation may facilitate myocyte fusion and formation ofmultinucleated myotubes. Another interesting point to note isthat STAT2 preferentially mediates the effect of JAK2 on theexpression of HGF, whereas both STAT2 and STAT3 regulateIGF2. This indicates that the roles of STAT2 andSTAT3are notcompletely redundant.In summary, we show here that JAK2, STAT2, and STAT3

positively regulate myogenic differentiation by regulating theexpression ofMyoD,MEF2,HGF, and IGF2. Our results furtherreinforce our previous notion that multiple members of theJAK/STAT family participate in myogenic differentiation viadistinct pathways and with distinct effects.

Acknowledgments—We thank Dr. Zilong Wen for various JAK andSTAT constructs and Carol Wong for technical assistance.

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